FIELD OF INVENTION
[0001] This inventive subject matter relates to a pharmaceutical useful in conferring passive
protection against diarrhea caused by enterotoxigenic
Esherichia coli.
BACKGROUND OF INVENTION
[0002] Enterotoxigenic
Escherichia coli (ETEC) are a principal cause of diarrhea in young children in resource-limited countries
and also travelers to these areas (1, 2). ETEC produce disease by adherence to small
intestinal epithelial cells and expression of a heat-labile (LT) and/or heat-stable
(ST) enterotoxin (3). ETEC typically attach to host cells via filamentous bacterial
surface structures known as colonization factors (CFs). More than 20 different CFs
have been described, a minority of which have been unequivocally incriminated in pathogenesis
(4).
[0003] Firm evidence for a pathogenic role exists for colonization factor antigen I (CFA/I),
the first human-specific ETEC CF to be described. CFA/I is the archetype of a family
of eight ETEC fimbriae that share genetic and biochemical features (5, 4, 6, 7). This
family includes coli surface antigen 1 (CS1), CS2, CS4, CS14, CS17, CS19 and putative
colonization factor 071 (PCFO71). The complete DNA sequences of the gene clusters
encoding CFA/1, CS1 and CS2 have been published (8, 9, 10, 11, 12). The genes for
the major subunit of two of the other related fimbriae have been reported (13, 6).
The four-gene bioassembly operons of CFA/I, CS1, and CS2 are similarly organized,
encoding (in order) a periplasmic chaperone, major fimbrial subunit, outer membrane
usher protein, and minor fimbrial subunit. CFA/I assembly takes place through the
alternate chaperone pathway, distinct from the classic chaperone-usher pathway of
type I fimbrial formation and that of other filamentous structures such as type IV
pili (14, 15). Based on the primary sequence of the major fimbrial subunit, CFA/I
and related fimbriae have been grouped as class 5 fimbriae (16).
[0004] Studies of CS1 have yielded details on the composition and functional features of
Class 5 fimbriae (17). The CS1 fimbrial stalk consists of repeating CooA major subunits.
The CooD minor subunit is allegedly localized to the fimbrial tip, comprises an extremely
small proportion of the fimbrial mass, and is required for initiation of fimbrial
formation (18). Contrary to earlier evidence suggesting that the major subunit mediates
binding (19), recent findings have implicated the minor subunit as the adhesin and
identified specific amino acid residues required for
in vitro adhesion of CS1 and CFA/I fimbriae (20). The inferred primary amino acid structure
of those major subunits that have been sequenced share extensive similarity. Serologic
cross-reactivity of native fimbriae is, however, limited, and the pattern of cross-reactivity
correlates with phylogenetically defined subtaxons of the major subunits (13).
[0005] Implication of the minor subunits of Class 5 fimbriae as the actual adhesins entreats
scrutiny regarding the degree of their conservation relative to that of the major
subunits. It was speculated that CooD and its homologs retained greater similarity
due to functional constraints imposed by ligand binding requirements and/or its immunorecessiveness,
itself attributable to the extremely large ratio of major to minor subunits in terms
of fimbrial composition. Studies were conducted to examine the evolutionary relationships
of the minor and major subunits of Class 5 ETEC fimbriae as well as the two assembly
proteins (21). It was demonstrated that evolutionary distinctions exist between the
Class 5 major and minor fimbrial subunits and that the minor subunits function as
adhesins. These findings provide practical implications for vaccine-related research.
[0006] The nucleotide sequence of the gene clusters that encode CS4, CS14, CS 17, CS19 and
PCFO71 was determined from wild type diarrhea-associated isolates of ETEC that tested
positive for each respective fimbria by monoclonal antibody-based detection (21).
The major subunit alleles of the newly sequenced CS4, CS14, CS17 and CS 19 gene clusters
each showed 99-100% nucleotide sequence identity with corresponding gene sequence(s)
previously deposited in GenBank, with no more than four nucleotide differences per
allele. Each locus had four open reading frames that encoded proteins with homology
to the CFA/I class chaperones, major subunits, ushers and minor subunits. As previously
reported (13), the one exception was for the CS14 gene cluster, which contained two
tandem open reading frames downstream of the chaperone gene. Their predicted protein
sequences share 94% amino acid identity with one another and are both homologous to
other Class 5 fimbriae major subunits.
[0007] Examination of the inferred amino acid sequences of all the protein homologs involved
in Class 5 fimbrial biogenesis reveals many basic similarities. Across genera, each
set of homologs generally share similar physicochemical properties in terms of polypeptide
length, mass, and theoretical isoelectric point. All of the involved proteins contain
an amino-terminal signal peptide that facilitates translocation to the periplasm via
the type II secretion pathway. None of the major subunit proteins contain any cysteine
residues, while the number and location of six cysteine residues are conserved for
all of the minor subunits except that of the
Y. pestis homolog 3802, which contains only four of these six residues.
[0008] Type 1 and P fimbriae have been useful models in elucidating the genetic and structural
details of fimbriae assembled by the classical chaperone-usher pathway (23,24, 25).
An outcome of this work has been development of the principle of donor strand complementation,
a process in which fimbrial subunits non-covalently interlock with adjoining subunits
by iterative intersubunit sharing of a critical, missing β-strand (22, 26). Evidence
has implicated this same mechanism in the folding and quaternary conformational integrity
of
Haemophilus influenzae hemagglutinating pili (28), and
Yersinia pestis capsular protein, a non-fimbrial protein polymer (29). Both of these structures are
distant Class I relatives of Type 1 and P fimbriae that are assembled by the classical
chaperone-usher pathway. From an evolutionary perspective, this suggests that the
mechanism of donor strand complementation arose in a primordial fimbrial system from
which existing fimbriae of this Class have derived. While donor strand complementation
represents a clever biologic solution to the problem of protein folding for noncovalently
linked, polymeric surface proteins, its exploitation by adhesive fimbriae other than
those of the classical usher-chaperone pathway has not been demonstrated.
[0009] Common to fimbriae assembled by the alternate chaperone pathway and the classical
chaperone-usher pathway are the requirement for a periplasmic chaperone to preclude
subunit misfolding and an usher protein that choreographs polymerization at the outer
membrane. That the fimbrial assembly and structural components of these distinct pathways
share no sequence similarity indicates that they have arisen through convergent evolutionary
paths. Nevertheless, computational analyses of the CFA/I structural subunits suggests
the possibility that donor strand complementation may also govern chaperone-subunit
and subunit-subunit interaction.
[0010] The eight ETEC Class 5 fimbriae clustered into three subclasses of three (CFA/I,
CS4, and CS14), four (CS1, PCFO71, CS17 and CS19), and one (CS2) member(s) (referred
to as subclasses 5a, 5b, and 5c, respectively) (21). Previous reports demonstrated
that ETEC bearing CFA/I, CS2, CS4, CS 14 and CS 19 manifest adherence to cultured
Caco-2 cells (6, 22). However, conflicting data have been published regarding which
of the component subunits of CFA/I and CS1 mediate adherence (19, 20).
[0011] This question of which fimbrial components is responsible for mediating adherence
was approached by assessing the adherence-inhibition activity of antibodies to intact
CFA/I fimbriae, CfaB (major subunit), and to non-overlapping amino-terminal (residues
23-211) and carboxy-terminal (residues 212-360) halves of CfaE (minor subunit) in
two different in vitro adherence models (21). It was demonstrated that the most important
domain for CFA/I adherence resides in the amino-terminal half of the adhesin CfaE
(21).
[0012] The studies briefly described above provide evidence that the minor subunits of CFA/I
and other Class 5 fimbriae are the receptor binding moiety (20,21). Consistent with
these observations, because of the low levels of sequence divergence of the minor
subunits observed within fimbrial subclasses 5a and 5b (20), the evolutionary relationships
correlated with cross-reactivity of antibodies against the amino-terminal half of
minor subunits representing each of these two subclasses (21). These studies strongly
suggest that the minor subunits of class 5 fimbriae are much more effective in inducing
antiadhesive immunity and thus an important immunogen for inducing protective antibody
(21).
[0013] Anti-diarrheal vaccines would be a preferable method of conferring protection against
diarrheal disease including ETEC caused diarrhea. However, because of the complexities
of constructing and licensing of effective vaccines other methods to confer interim
protection have been sought. A measure shown to hold considerable promise in the prevention
of diarrhea is passive, oral administration of immunoglobulins against diarrhea causing
enteropathogens. Products with activity against ETEC,
Shigella, and rotavirus have been shown to prevent diarrhea in controlled studies with anti-cryptosporidial
bovine milk immunoglobulins (BIgG) preparations (30 - 33). Freedman, et al. (33) utilizes
isolated CFA from ETEC bacteria. Furthermore, favorable encouraging results have been
observed using this approach with anti-cryptosporidial BIgG preparations (34, 35).
[0014] Accordingly, an object of this invention is an immunoglobulin supplement capable
of providing prophylactic protection against ETEC infection. Because the minor subunit
(adhesin) is the fimbrial component directly responsible for adherence, administration
of anti-adhesin antibodies will likely confer significantly greater protection than
antibodies raised against intact fimbriae or intact bacteria. Furthermore, another
object of the invention is a method for the production of passive prophylactic formulation
against ETEC, containing anti-adhesin immunoglobulin. The use of recombinant minor
fimbrial subunit polypeptides in the immunoglobulin production method will provide
enhanced antibody yields and standardization over the use of intact fimbriae or whole
cells.
SUMMARY OF INVENTION
[0015] The invention provides a method of producing a pharmaceutical composition, a pharmaceutical
composition and a use of a pharmaceutical composition according to claim 1.
[0016] Vaccines are the preferred method for conferring anti-diarrhea protection in potentially
exposed populations. However, there are no currently licensed effective vaccines against
ETEC. Therefore, an interim protective measure, until vaccines can be developed, is
the administration of oral passive protection in the form of anti-adhesin immunoglobulin
supplements derived from bovine, or other milk producing animal, colostrum or milk.
[0017] An object of the invention is a anti-
Escherichia coli antibody prophylactic formulation that is specific to class 5 enterotoxigenic
E. coli fimbriae adhesin.
[0018] Another object of the invention is a method for conferring passive immunity using
an anti-
E.
coli antibody prophylactic formulation that is specific to class five
Escherichia coli fimbriae adhesin including CfaE and CsbD.
[0019] An additional object of the invention is a method of conferring passive immunity
to enterotoxigenic
E. coli by administering a food supplement containing anti-
E.
coli antibody specific to Class 5 fimbriae adhesins.
[0020] A still further object of the invention is a method of producing an anti-
E.
coli adhesin milk antibody by administering recombinant adhesin polypeptides to domestic
animals such as cows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021]
FIG. 1. A highly conserved β-strand motif in the major structural subunits of Class
5 fimbriae. This is a multiple alignment of the amino-termini of the mature form of
the major subunits, with consensus sequence shown below. This span is predicted to
form an interrupted β-strand motif spanning residues 5-19 (demarcated by yellow arrows
below consensus). Shading of conserved residues indicates class as follows: blue,
hydrophobic; red, negatively charged residues; turquoise, positively charged residues;
and green, proline. Also shown are the sequence identification numbers (SEQ ID No.)
for the associated polypeptides. Abbrevations: Bcep, Burkholderia cepacia; Styp, Salmonella typhi. U, hydrophobic residue; x, any residue; Z, E or Q.
FIG. 2. Panel A, Schematic diagram showing the domains of independent CfaE variant
constructs with C-terminal extensions comprising the N-terminal ß-strand span of CfaB
varying in length from 10 to 19 residues. Each construct contains a short flexible
linker peptide (DNKQ) intercalated between the C-terminus of the native CfaE sequence
and the donor ß-strand. The vertical arrow identifies the donor strand valine that
was modified to either a proline (V7P) to disrupt the secondary ß-strand motif. Panel
B, Western blot analysis of periplasmic concentrates from the series of strains engineered
to express CfaE and the variants complemented in cis with varying lengths of the amino-terminal span of mature CfaB. The primary antibody
preparations used were polyclonal rabbit antibody against CfaE. Lanes correspond to
preparations from the following constructs: Lane 1, dsc10CfaE; 2, dsc11CfaE; 3, dsc12CfaE; 4, dsc13CfaE; 5, dsc13CfaE[V7P]; 6, dsc14CfaE; 7, dsc16CfaE; 8, dsc19CfaE; and 9, CfaE. Molecular weight markers (in kD) are shown to the left. Panel C,
schematic representation of the engineered components of dsc19CfaE(His)6, containing the native CfaE sequence (including its Sec-dependent N-terminal
signal sequence), with an extension at its C-terminus consisting of a short linker
sequence (i.e., DNKQ), the 19 residue donor strand from the N-terminus of mature CfaB,
and a terminal hexahistidine affinity tag.
FIG. 3. Reactivity of products with a panel of CFA/I-related antigens by ELISA.
FIG. 4. In vitro functional activity of antibodies to BIgG anti-CfaE.
FIG. 5. Reactivity of products with a panel of CS17-related antigens by ELISA.
FIG. 6. In vitro functional activity of BIgG anti-CsbD.
DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS
[0022] Vaccines are the preferred prophylactic measure for long-term protection against
ETEC caused diarrhea. However, development of effective vaccines is typically difficult
and time-intensive. Furthermore, even after an effective ETEC vaccine is developed,
protection against ETEC caused disease is not conferred until an adequate dose regimen
is completed. Therefore, there is a need for effective, safe and easy to take passive
prophylactic measures. A particularly promising approach, for example, is the use
of bovine milk immunoglobulins (BIgG) preparations (30-33).
[0023] Computational analyses of the CFA/I structural subunits suggest that donor strand
complementation governs chaperone-subunit and subunit-subunit interaction. The major
subunits of Class 5 fimbriae share a highly conserved amino-terminal span predicted
to form a β strand (FIG 1). Based on its predicted structure and location, the β-strand-like
structure is donated to neighboring major subunit (e.g. CfaB) along the alpha-helical
stalk and to an adhesin (e.g. CfaE) at the fimbrial tip. The highly conserved nature
of the amino-terminal β strand of CfaB and its homologs, together with the precedent
that the amino-terminus of type 1 fimbrial subunits functions as the exchanged donor
strand in filament assembly suggested this as a good candidate for the donor β strand
that noncovalently interlocks CFA/I subunits.
[0024] ETEC fimbriae are classified based on genetic and structural analysis and many fimbriae
associated with disease fall into the Class 5 fimbrial grouping, which includes CFA/I,
CS17 and CS2. Class 5 fimbriae adhesins each share significant characteristics that
clearly differentiate these members as belonging to a recognizable genus. Although
Class 5 fimbriae are distinguishable serologically, they share similar architecture
in that they are composed of a major stalk forming subunit (e.g., CfaB of CFA/I) and
a minor tip-localized subunit (e.g., CfaE of CFA/I) that we have found serves as the
intestinal adhesin. A comparison of amino acid sequences of the major and minor subunits
(i.e., fimbrial adhesins) clearly show a strong amino acid sequence relatedness as
well as sequence homology, as illustrated in Table 1, for the major subunits and Table
2 for adhesin molecules. In Table 1 and 2 the shaded areas show similarity of residues
and the unshaded areas show residue identity. As illustrated in Table 2, the fimbrial
adhesins display, as well as the major subunits, a high level of residue identity,
ranging from 47% to 98%. Additionally, fimbrial adhesins have significant amino acid
sequence conservation, including a conserved structural motif in the carboxy-terminal
domain of both the major and minor subunits (i.e., the beta-zipper motif). This structure
indicates that the C-terminal domain of these proteins are involved in subunit-subunit
interaction.
Table 1
|
CfaB |
CooA |
CotA |
CsfA |
CsuA1 |
CsbA |
CsdA |
CosA |
CfaB |
- |
.53 |
.51 |
.66 |
.58 |
.50 |
.52 |
.52 |
CooA |
.74 |
- |
.50 |
.57 |
.51 |
.61 |
.59 |
.90 |
CotA |
.67 |
.71 |
- |
.50 |
.52 |
.45 |
.47 |
.52 |
CsfA |
.81 |
.75 |
.71 |
- |
.62 |
.54 |
.55 |
.56 |
CsuA1 |
.75 |
.72 |
.71 |
.78 |
- |
.51 |
.50 |
.52 |
CsbA |
.73 |
.76 |
.67 |
.72 |
.73 |
- |
.88 |
.61 |
CsdA |
.72 |
.75 |
.69 |
.71 |
.72 |
.92 |
- |
.57 |
CosA |
.71 |
.95 |
.74 |
.73 |
.73 |
.79 |
.78 |
- |
3 letter codes: CFA/I, Cfa; CS1, Coo; CS2, Cot; CS4, Csf; CS14, Csu; CS17, Csb; CS19,
Csd; PCFO71, Cos. |
Table 2
|
CfaE |
CooD |
CotD |
CsfD |
CsuD |
CsbD |
CsdD |
CosD |
CfaE |
- |
.51 |
.46 |
.80 |
.82 |
.51 |
.51 |
.50 |
CooD |
.65 |
- |
.49 |
.51 |
.50 |
.97 |
.97 |
.98 |
CotD |
.64 |
.63 |
- |
.47 |
.48 |
.48 |
.48 |
.48 |
CsfD |
.87 |
.65 |
.64 |
- |
.94 |
.50 |
.50 |
.50 |
CsuD |
.88 |
.64 |
.65 |
.97 |
- |
.50 |
.50 |
.50 |
CsbD |
.66 |
.97 |
.62 |
.65 |
.65 |
- |
.97 |
.96 |
CsdD |
.66 |
.97 |
.63 |
.64 |
.65 |
.97 |
- |
.98 |
CosD |
.65 |
.99 |
.62 |
.64 |
.65 |
.96 |
.97 |
- |
3 letter codes: CFA/I, Cfa; CS1, Coo; CS2, Cot; CS4, Csf; CS14, Csu; CS17, Csb; CS19,
Csd; PCFO71, Cos. |
[0025] Toward the development of an ETEC antigen, we constructed a conformationally-stable
construct wherein an amino-terminal donor β-strand of CfaB provides an
in cis carboxy-terminal extension of CfaE to confer conformational stability and protease
resistance to this molecule, forming a soluble monomer capable of binding human erythrocytes.
In order to identify common structural motifs, multiple alignments of the amino acid
sequences of the eight homologs of the major and minor subunits of Class 5 ETEC fimbriae
were generated. Secondary structure prediction algorithms indicated that both subunits
form an amphipathic structure rich in β-strands distributed along their length. Twenty
six percent of the consensus minor subunit sequence is predicted to fold into a β-confirmation,
comprising 17 interspersed β strands, which might be expected to form a hydrophobic
core. Sakellaris
et al have previously suggested that an amino acid span forms a β-zipper motif, analogous
to that of class I fimbrial subunits, that plays a role in fimbrial subunit-chaperone
interaction (27).
[0026] The following example discloses the production of a CfaE immunogen using a donor
strand from CfaB. However, one of skill in the art, following this disclosure, would
be able to engineer constructs to serve as an immunogen using donor strands from other
class 5 major subunits in conjunction with other adhesin constructs, such as CsbD,
CsfD, CsuD, CooD, CosD, CsdD, and CotD. The major Class 5 fimbrial subunits are listed
in Table 3 along with the corresponding SEQ ID No. corresponding to the subunit's
amino acid sequence donor strand. Table 4 lists the amino acid sequence of the Class
5 adhesin and their respective SEQ ID No.
Table 3
Major Subunit |
SEQ ID No.of Donor Strand Amino Acid Sequence |
CfaB |
1 |
CsfA |
2 |
CsuA1 |
3 |
CsuA2 |
4 |
CooA |
5 |
CosA |
6 |
CsbA |
7 |
CsdA |
8 |
CotA |
9 |
Table 4
Minor Subunit |
SEQ ID. No. of Amino Acid Sequence |
CfaE |
11 |
CsbD |
22 |
CsfD |
27 |
CsuD |
28 |
CooD |
29 |
CosD |
30 |
CsdD |
31 |
CotD |
32 |
[0027] An aspect of this invention is a method for the production of a passive prophylactic
against Class 5 fimbrial adhesin of ETEC bacteria. Examples using specific Class 5
fimbrial adhesins are provided in order to illustrate the invention. However, other
Class 5 fimbrial adhesins, and their associated major subunits can also be utilized
by one of skill in the art
Example 1: Production of anti-CfaE bovine immunoglobulin
[0028] As mentioned above, the highly conserved nature of the amino-terminal β strand of
CfaB and its homologs, together with other structure/function studies in type 1 fimbrial
subunits, suggested this structure as a good candidate for the donor P strand that
interlocks CFA/I subunits. In order to test this hypothesis with respect to the minor
adhesive subunit, a plasmid was engineered to express a CfaE variant containing a
C-terminal extension consisting of a flexible hairpin linker (DNKQ, SEQ ID No. 10)
followed by an amino acid sequence of CfaB (FIG 2). It was found that a CfaB donor
strand length of at least 12 to as many as 19 amino acids was necessary to obtain
a measurable recovery of CfaE. In studies using constructs containing a 12 to 19 amino
acid donor strand, where mutations were introduced to break the β strand, it was demonstrated
that the β strand is important to the observed stability achieved by the C-terminal
amino acid extension. It was further determined that the C-terminal β strand contributed
by CfaB in
cis precludes chaperone (e.g. CfaA)-adhesin complex formation.
[0029] In this example, a recombinant CfaE antigen was constructed, as shown in Figure 2C,
by fusing a Cfa E polypeptide sequence (SEQ ID No. 11), encoded by the nucleotide
sequence of SEQ ID No. 18 to the N-terminal amino acid sequence of a linker polypeptide
(SEQ ID No. 10) which is in-turn linked at its C-terminus to a 19 amino acid CfaB
donor strand corresponding to amino acids 1-19 of SEQ ID No. 1. Although, SEQ ID No.
10 was utilized for a linker, other amino acid sequences have been found acceptable,
including SEQ ID No. 12 and 13. For this example, the CfaB major subunit donor strand
used is shown in SEQ ID No. 1 which is encoded by the nucleotide sequence of SEQ ID
No. 20. However, based on the observation that the a donor strand of 12 to 19 amino
acids is suitable for significant CfaE recovery, a recombinant antigen containing
12 to 19 amino acids can be utilized. Similarly, recombinant peptides can be constructed
containing all or a portion of SEQ ID No. 11 as long as the amino acid sequence contains
anti-CfaE B-cell epitopes.
[0030] The CfaE construct containing the 19 amino acid major subunit donor strand was constructed
by first inserting
cfaE into plasmid vectors by in vitro recombination using the Gateway
® system (Invitrogen, Carlsbad, CA). Primers with the following sequences were used
for the initial cloning into pDONR207™: dsc-CfaE 13-1 (forward), 5'-TCG ACA ATA AAC
AAG TAG AGA AAA ATA TTA CTG TAA CAG CTA GTG TTG ATC CTT AGC-3' (SEQ ID No. 14); and
dsc-CfaE 13-2 (reverse), 5'-TCG AGC TAA GGA TCA ACA CTA GCT GTT ACA GTA ATA TTT TTC
TCT ACT TGT TTA TTG-3' (SEQ ID No 15). The PCR products flanked by
attB recombination sites were cloned into the donor vector pDONR201™ (Gateway
® Technology, Invitrogen, Carlsbad, CA), using the Gateway BP
® reaction to generate the entry vector pRA13.3. In the Gateway LR
® reaction the gene sequence was further subcloned from pRA13.3 into the modified expression
vector pDEST14-Kn
r (vector for native expression from a T7 promoter) to generate the plasmid pRA14.2.
The pDEST14-Kn
r vector was constructed by modifying pDEST14
® (Gateway
® Technology, Invitrogen, Carlsbad, CA) by replacement of ampicillin with kanamycin
resistance. The presence of the correct
cfaE was confirmed by sequence analysis.
E. coli strain BL21SI™ (Invitrogen, Carlsbad, CA) was used for the expression of the pRA14.1
and related CfaE donor strand complemented constructs.
[0031] The above procedure was utilized to construct a CfaE/donor strand recombinant construct.
However, constructs containing other adhesin molecules can also be constructed, including
the minor subunits: CsfD, CsuD, CooD, CosD, CsdD, CsbD and CotD, in conjunction with
the appropriate donor strand from the major subunits as listed in Table 1. For example,
a recombinant CsbD construct was designed comprising a CsbD polypeptide sequence comprising
all or a portion of SEQ ID No. 22 fused at the C-terminal end, via a linker polypeptide
of SEQ ID No 10, to a CsbA major subunit donor strand of a polypeptide sequence SEQ
ID No. 6 that is encoded by the nucleotide sequence of SEQ ID No. 21.
Development of pET/adhesin construct for large scale antigen production
[0032] The DNA construct encoding dsc
19CfaE was then excised from pDEST14
® vector and inserted into pET24(a)™ in order to encode a variant CfaE construct that
incorporates a carboxy-terminal polyhistine tail after the CfaB donor strand. This
construct, with a polypeptide sequence of SEQ ID No 23 is designated dsc
19CfaE(His)
6 and is encoded by the nucleotide sequence of SEQ ID No. 24.
[0033] Construction of the dsc
19cfaE insert was carried out by amplifying the pDEST 14 vector by polymerase chain
reaction using a NdeI containing forward primer and an XhoI containing reverse primer,
SEQ ID No 16 and 17, respectively. The dsc
19cfaE coding region was directionally ligated into an NdeI/XhoI restricted pET24a plasmid.
The insert containing pET24a™ plasmid was used to transform NovaBlue-3™ BL21 (EMD
Biosciences, Novagen
® Brand, Madison, WI) bacteria. Transformed colonies were then selected and re-cultured
in order to expand the plasmid containing bacteria. Plasmid inserts from selected
colonies were then sequenced. These plasmids were then reinserted into BL 21 (DE3)
(EMD Biosciences, Novagen
® Brand, Madison, WI) competent cells and the DNA insert sequence confirmed.
[0034] Similar to the method used to construct dsc
19CfaE(His)
6, a DNA construct encoding dsc
19CsbD was also made by insertion of CsbD and a CsbA donor strain sequence into pET24a™.
This construct has a polypeptide sequence of SEQ ID No. 25 and is encoded by the nucleotide
sequence of SEQ ID No. 26. The donor strand sequence from CsbA used in designing the
construct is disclosed as SEQ ID No. 6. Like the CfaE construct, the 19 amino acid
sequence from CsbA corresponding to amino acids 1-19 of SEQ ID No. 6 was used. However
donor strand sequences ranging from the 12 to 19 amino acids can be used.
Production of dsc19CfaE(His)6.
[0035] A number of growth conditions and media can be utilized for large-scale production
of the dsc
19CfaE(His)
6, or other adhesin/donor strand construct. For example initiation of culture can be
conducted using 1.0 µM to 1.0 mM isopropyl-β-D-thiogalactopyranosid (IPTG) at an induction
temperature of 32° C to 25° C for 1 to 4 hours. In this example, LB media was utilized
with a 1.0 µM IPTG concentration at 32° C for 3 hours. However, APS™ and other media
formulations can also be used. The dsc
19CfaE(His)
6, or other recombinant adhesin construct, is purified on a Ni column. Yield of construct
is at least 0.45 to 0.9 mg of protein/L of culture.
Manufacture of BIgG
[0036] Antibody to recombinant antigen is produced in the colostrum or milk of domesticated
cattle, including Holsteins. A total of three intramuscular vaccinations each in a
volume of two ml containing 500 µg of antigen each is administered at a single site.
Vaccinations are given approximately three weeks apart with the final vaccination
1 to 2 weeks prior to calving. At calving the first four milkings are collected, the
volume estimated and a sample tested for anti-adhesin antibody by enzyme-linked immunosorbent
assay (ELISA). FIG 3 shows the reactivity of anti-CFA/I BIgG and anti-CfaE BIgG products.
CFA/I BIgG gives a higher level of reactivity to CFA/I antigen than anti-CfaE by ELISA
(FIG. 3A). This is due to the fact that CFA/I antigen used to coat the ELISA plate
is made of primarily the CfaB major subunit and the CfaE minor subunit is present
as a minor component only. As expected, the anti-CfaE BIgG product has a much stronger
reaction with CfaE compared to either AEMI or anti-CFA/I BIgG (FIG. 3B). This confirms
that immunization of cows with the CfaE antigen greatly enhances the generation of
antibodies to adhesin, CfaE.
[0037] Further processing of the collected product can be undertaken. For example, frozen
milk is fractionated to remove caseins through a cheese-making step. The whey fraction,
containing most immunoglobulins is then drained from the cheese curd and pasteurized
under standard dairy conditions. The immunoglobulin-enriched whey fraction is then
concentrated and residual milk fat is removed by centrifugation at room temperature.
Subsequently, phospholipid and non-immunoglobulin proteins can be removed (36). The
final product is then concentrated to 15-20% solids and salts removed by continuous
diafiltration against three buffer changes. The final product is then tested for by
ELISA.
[0038] In addition to the characterization of antibody reactivity of BIgG to ETEC antigens,
the functional activity of the antibodies was evaluated. As the receptor(s) for CFA/I
is not defined, a surrogate assay for adhesion of ETEC to target cells
in vitro was used. ETEC expressing certain fimbriae (including CFA/I) adhere to and agglutinate
human and/or bovine erythrocytes in a mannose-resistant hemagglutination assay (MRHA).
This is used as a surrogate marker for adhesion of ETEC whole cells, fimbriae or purified
minor subunits of fimbriae to target eukaryotic epithelial cells. This phenomenon,
described as hemagglutination inhibition (HAI), is an indicator of antibodies capable
of neutralizing adhesion of ETEC to target cells.
[0039] In FIG. 4, human erythrocytes were agglutinated by ETEC expressing CFA/I, CS4 or
CS 14 in a mannose-resistant manner (MRHA). This MRHA can be inhibited by pre-incubation
of bacteria with anti-CFA/I BIgG or anti-CfaE BIgG. Shown in FIG. 4, both anti-CFA/I
BIgG and anti-CfaE BIgG contained antibodies capable of inhibiting the ability of
ETEC that express the homologous fimbriae from agglutinating human erythrocytes. FIG.
4 shows the titer of BIgG (expressed as mg IgG/ml) required to neutralized aggluntination
of bovine erythrocytes by ETEC expressing different colonization factors. The concentrations
of BIgG products tested were adjusted so the minimal concentrations of IgG were equal
in both products. Therefore, the data is expressed as the concentration of IgG that
is required to inhibit MRHA by ETEC expressing CFA/I, CS4 or CS14 fimbriae. As little
as 14 to 17 µg/ml of bovine IgG present in the BIgG powders are required
in vitro to inhibit MRHA.
[0040] Strong inhibitory activity is provided by anti-CFA/I, as expected, with an equivalent
level of inhibition provide by anti-CfaE. Of importance is that both anti-CFA/I and
anti-CfaE show cross-reactivity of binding inhibition against CS4 and CS 14. This
illustrates that an anti-CfaE prophylactic antibody will have utility in conferring
protection against other related antigens.
Example 2: Production of anti-CfaD (CS17) bovine immunoglobulin
[0041] Use of other class 5 fimbrial adhesins are also contemplated as eliciting protective
passive antibody production. As a further illustration, results of inhibition by antibody
to CS 17 (i.e., CsbD) is presented in FIG. 5. The antigen used to elicit antibody
was a CsbD polypeptide (SEQ ID No. 22) expressing construct. The construct was engineered
similar to that for CfaE, in Example 1, above but with a nucleotide sequence encoding
CsbD (SEQ ID No. 19). The construct was designated dsc
19CsbD[His]
6. The donor strand consisted of 19 amino acids of CsbA (SEQ ID No. 7).
[0042] As can be seen in FIG. 5, like that for CfaE, antibody to CsbD was highly efficient
at inhibiting MRHA. Also, like that observed for CfaE, anti-CsbD antibody also afforded
cross-protection against CS4 and CS2.
[0043] The functional activity of BIgG to CS 17 and CsbD was also evaluated, as in FIG.
4. These results are illustrated in FIG. 6. Like that observed for anti-CfaE and anti-CFA/I,
BIgG against both CS 17 and CsbD exhibited significant inhibitory activity. However,
more pronounce than for anti-CfaE BIgG, anti-CsbD, compared to anti-CS17 BIgG, exhibited
significant inhibitory activity even to heterologous antigens. These observations,
along with that observed for CfaE indicate that only a limited number of species within
the Class five adhesin genus is likely to be required for efficacious passive protection.
Example 3: Specific regions of ETEC fimbrial adhesin are important for immunoreactivity
and stability
[0044] Crystollgraphic analysis of the dscCfaE reveals that fimbrial adhesin is composed
of two domains, an adhesin domain, formed by the amino-terminal segment of the adhesin
molecule and a C-terminal pilin domain. The two domains are separated by a three amino
acid linker. In an attempt to understand those regions of fimbrial adhesin, amino
acid substitutions where made and the ensuing immunoreactivity analyzed. It was found
that replacement of arginine 67 or arginine 181 with alanine, on CfaE abolishes the
in vitro adherence phenotype of the molecule. These amino acids positions are located on exposed
regions of the molecule with residue Arg 181 located on the distal portion of the
amino-terminus of the domain. Therefore, this region of CfaE and the comparable region
of the other fimbrial adhesins, is important for efficacious immune induction. Table
3 summarizes the positions in the eight adhesins. Also shown in Table 3 is that region
of the domain that has added importance, based on crystollgraphic analysis, in conferring
structural stability of the fimbrial adhesin molecule.
Table 3
Fimbrial Adhesin |
Fimbrial Adhesin domain residues important for immunoreactivity |
Fimbrial Adhesin domain residues important for structural stability |
CfaE |
amino acids 66-183 |
amino acids 22-202 |
CsuD |
amino acids 66-183 |
amino acids 22-202 |
CsfD |
amino acids 66-183 |
amino acids 22-202 |
CooD |
amino acids 65-183 |
amino acids 20-205 |
CosD |
amino acids 65-185 |
amino acids 20-205 |
CsbD |
amino acids 65-183 |
amino acids 20-205 |
CsdB |
amino acids 65-183 |
amino acids 20-205 |
CotD |
amino acids 58-177 |
amino acids 14-196 |
[0045] Stabilization of the adhesin domain of intact fimbrial adhesin molecules is provided
by the major subunit. However, devoid of the pili domain, fimbrial adhesin exhibits
greater conformational stability than the intact molecule with concomitant retention
of immunoreactivity. As an alternative to administration of the intact adhesin molecule,
administration of only the adhesin domain is an alternative immunogen for induction
of anti-fimbrial adhesin antibodies. Therefore, as an example, recombinant adhesin
domain constructs encoding CfaE, CsbD and CotD adhesin domains, but not containing
the pili domain, were constructed, by polymerase chain reaction amplification of the
adhesin domain and inserted into pET 24a™. The amino acid sequences of the recombinant
product is illustrated in SEQ ID No.s 35, 36 and 37. Incorporation of a polyhistidine
tail, as in Example 1 and 2, facilitates purification of the ensuing expressed product.
Example 4: Administration of anti-fimbrial adhesin as prophylactic against ETEC
[0046] Class five fimbrial adhesins can be used for the development of prophylactic protection
against ETEC infection. Protection is provided by collecting colostrums or milk product
from fimbrial adhesin, either native or recombinant
Escherichia coli adhesin, immunizing cows. Immunization can be by any number of methods. However,
a best mode is the administration of three doses intramuscularly three weeks apart
with a final administration, 1 to 2 weeks prior to calving, of se in 1 to 2 ml volume
containing up to 500 µg of said adhesin. Collection of milk or colostrums can be at
anytime, however optimal results likely is when collection is 1 to 2 weeks prior to
calving.
[0047] Administration of the anti-adhesin bovine immunoglobulin as a prophylactic is achieved
by ingestion of 0.1g IgG/dose to 20.0 g of IgG/dose. The anti-adhesin bovine colostrum
or milk immunoglobulin can be ingested alone or mixed with a number of beverages or
foods, such as in candy. The immunglobulin can also be reduced to tablet or capusular
form and ingested.
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SEQUENCE LISTING
[0049]
<110> Naval Medical Research Center Savarino, Stephen
<120> Anti-Adhesin based passive prophylactic
<130> NC 97,088
<160> 37
<170> PatentIn version 3.3
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1. A method of producing a pharmaceutical composition which is suitable as a passive
immunoprophylactic against enterotoxigenic
Escherichia coli, the method comprising the steps:
a. administering to a milk producing domesticated non-human animal an immunogen composed
of one or more Class five Escherichia coli fimbrial adhesions, each of the one or more fimbrial adhesins being linked at its
C-terminus to a linker which is operatively linked at its C-terminus to an Escherichia coli major fimbrial subunit, wherein said linker is composed of an amino acid sequence
selected from the group consisting of SEQ ID No. 10, SEQ ID No. 12, and SEQ ID No.13;
and
b. collecting anti-adhesin immunoglobulin containing colostrums or milk from said
domesticated non-human animal.
2. The method of claim 1, wherein the concentration of said anti-adhesin immunoglobulin
in said collected colostrums or milk is adjusted to 0.1 g IgG per dose to 20.0 g of
IgG per dose.
3. The method of claim 1, wherein said domesticated animal is a cow or goat.
4. The method of claim 1, wherein said fimbrial adhesin is selected from the group consisting
of CfaE, CsfD, CsuD, CooD, CosD, CsdD, CsbD and CotD.
5. The method of claim 1, wherein said Escherichia coli major fimbrial subunit is selected from the group consisting of CfaB, CsfA, CsuA1,
CsuA2, CooA, CosA, CsbA, CsdA and CotA.
6. The method of claim 4, wherein said immunogen is an Escherichia coli fimbrial adhesion domain and polyhistidine tail fusion polypeptide composed of the
amino acid sequence selected from the group consisting of SEQ ID No. 35, SEQ ID No.
36 and SEQ ID No. 37.
7. The method of claim 1, wherein said Escherichia coli fimbrial adhesin is a monomer or polymer of adhesin polypeptides.
8. The method of claim 1, wherein said immunogen contains a polyhistidine tail linked
at the C-terminus of said Escherichia coli major fimbrial subunit.
9. The method of claim 1, wherein said fimbrial adhesin is an amino acid sequence selected
from the group consisting of SEQ ID No. 11, SEQ ID No. 22, SEQ ID No. 27, SEQ ID No.
28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32 and fragments thereof.
10. The method of claim 4, wherein CfaE is composed of the amino acid sequence of SEQ
ID No. 11 encoded by all or a portion of the nucleotide sequence of SEQ ID No. 18
or fragment thereof.
11. The method of claim 4, wherein said CsbD is composed of the amino acid sequence SEQ
ID No. 22 encoded by the nucleotide sequence of SEQ ID No. 19 or fragment thereof.
12. The method of claim 4, wherein said CotD is composed of the amino acid sequence of
SEQ ID No. 32 or fragment thereof.
13. The method of claim 1, wherein said Escherichia coli fimbrial adhesin is composed of amino acids 58-185 or a sequence selected from the
group consisting of SEQ ID No. 11, SEQ ID No. 22, SEQ ID No. 27, SEQ ID No. 28, SEQ
ID No. 29, SEQ ID No. 30, SEQ ID No. 31, and SEQ ID No. 32.
14. The method of claim 1, wherein said Escherichia coli fimbrial adhesin is composed of amino acids 14-205 or a sequence selected from the
group consisting of SEQ ID No. 11, SEQ ID No. 22, SEQ ID No. 27, SEQ ID No. 28, SEQ
ID No. 29, SEQ ID No. 30, SEQ ID No. 31, and SEQ ID No. 32.
15. The method of claim 1, wherein said major fimbrial subunit is an amino acid sequence
selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ
ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, and SEQ ID No. 9.
16. The method of claim 1, wherein said immunogen is a fusion polypeptide containing a
polyhistidine tail composed of the amino acid sequence selected from the group consisting
of SEQ ID No. 24 encoded by nucleotide SEQ ID No. 23, SEQ ID No. 26, encoded by SEQ
ID No. 25 and SEQ ID No. 35.
17. The method of claim 1, wherein said major fimbrial subunit is CfaB with a polypeptide
sequence of SEQ ID No. 1 encoded by nucleotide sequence SEQ ID No. 20.
18. The method of claim 1, wherein said major fimbrial subunit is CsbA with a polypeptide
sequence of SEQ ID No. 7 encoded by nucleotide sequence SEQ ID No. 21.
19. The method of claim 1, wherein said major fimbrial subunit is CotA with a polypeptide
sequence of SEQ ID No. 9.
1. Verfahren zur Herstellung einer pharmazeutischen Zusammensetzung, welche als eine
passive Immunprophylaxe gegen enterotoxigene
Escherichia coli geeignet ist, wobei das Verfahren folgende Schritte umfasst:
a. Verabreichen eines Immunogens zusammengesetzt aus einem oder mehreren Escherichia coli Fimbrienadhäsinen der Klasse Fünf, wobei jedes des/der einen oder mehreren Fimbrienadhäsine
an seinem C-Terminus mit einem Linker verknüpft ist, welcher wirkungsmäßig an seinem
C-Terminus mit einer Escherichia coli Hauptfimbrien-Untereinheit verknüpft ist, wobei der Linker aus einer Aminosäuresequenz
ausgewählt aus der Gruppe bestehend aus SEQ ID NR. 10, SEQ ID NR. 12 und SEQ ID NR.
13 zusammengesetzt ist, an ein milcherzeugendes domestiziertes nichthumanes Tier;
und
b. Sammeln des Antiadhäsin-Immunglobulins, welches Kolostrum oder Milch von dem domestizierten
nichthumanen Tier enthält.
2. Verfahren nach Anspruch 1, wobei die Konzentration des Antiadhäsin-Immunglobulins
in dem/der gesammelten Kolostrum oder Milch auf 0,1 g IgG pro Dosis bis 20,0 g IgG
pro Dosis angepasst ist.
3. Verfahren nach Anspruch 1, wobei das domestizierte Tier eine Kuh oder Ziege ist.
4. Verfahren nach Anspruch 1, wobei das Fimbrienadhäsin ausgewählt ist aus der Gruppe
bestehend aus CfaE, CsfD, CsuD, CooD, CosD, CsdD, CsbD und CotD.
5. Verfahren nach Anspruch 1, wobei die Escherichia coli Hauptfimbrien-Untereinheit ausgewählt ist aus der Gruppe bestehend aus CfaB, CsfA,
CsuA1, CsuA2, CooA, CosA, CsbA, CsdA und CotA.
6. Verfahren nach Anspruch 4, wobei das Immunogen eine Escherichia coli Fimbrien-Adhäsionsdomäne und ein Polyhistidinschwanz-Fusionspolypeptid zusammengesetzt
aus der Aminosäuresequenz ausgewählt aus der Gruppe bestehend aus SEQ ID NR. 35, SEQ
ID NR. 36 und SEQ ID NR. 37 ist.
7. Verfahren nach Anspruch 1, wobei das Escherichia coli Fimbrienadhäsin ein Monomer oder Polymer von Adhäsin-Polypeptiden ist.
8. Verfahren nach Anspruch 1, wobei das Immunogen einen Polyhistidinschwanz verknüpft
am C-Terminus der Escherichia coli Hauptfimbrien-Untereinheit enthält.
9. Verfahren nach Anspruch 1, wobei das Fimbrienadhäsin eine Aminosäuresequenz ausgewählt
aus der Gruppe bestehend aus SEQ ID NR. 11, SEQ ID NR. 22, SEQ ID NR. 27, SEQ ID NR.
28, SEQ ID NR. 29, SEQ ID NR. 30, SEQ ID NR. 31, SEQ ID NR. 32 und Fragmenten davon
ist.
10. Verfahren nach Anspruch 4, wobei CfaE zusammengesetzt ist aus der Aminosäuresequenz
von SEQ ID NR. 11 codiert durch die gesamte oder einen Abschnitt der Nukleotidsequenz
von SEQ ID NR. 18 oder ein Fragment davon.
11. Verfahren nach Anspruch 4, wobei das CsbD zusammengesetzt ist aus der Aminosäuresequenz
von SEQ ID NR. 22 codiert durch die Nukleotidsequenz von SEQ ID NR. 19 oder ein Fragment
davon.
12. Verfahren nach Anspruch 4, wobei das CotD zusammengesetzt ist aus der Aminosäuresequenz
von SEQ ID NR. 32 oder ein Fragment davon.
13. Verfahren nach Anspruch 1, wobei das Escherichia coli Fimbrienadhäsin zusammengesetzt ist aus den Aminosäuren 58-185 oder einer Sequenz
ausgewählt aus der Gruppe bestehend aus SEQ ID NR. 11, SEQ ID NR. 22, SEQ ID NR. 27,
SEQ ID NR. 28, SEQ ID NR. 29, SEQ ID NR. 30, SEQ ID NR. 31 und SEQ ID NR. 32.
14. Verfahren nach Anspruch 1, wobei das Escherichia coli Fimbrienadhäsin zusammengesetzt ist aus den Aminosäuren 14-205 oder einer Sequenz
ausgewählt aus der Gruppe bestehend aus SEQ ID NR. 11, SEQ ID NR. 22, SEQ ID NR. 27,
SEQ ID NR. 28, SEQ ID NR. 29, SEQ ID NR. 30, SEQ ID NR. 31 und SEQ ID NR. 32.
15. Verfahren nach Anspruch 1, wobei die Hauptfimbrien-Untereinheit eine Aminosäuresequenz
ausgewählt aus der Gruppe bestehend aus SEQ ID NR. 1, SEQ ID NR. 2, SEQ ID NR. 3,
SEQ ID NR. 4, SEQ ID NR. 5, SEQ ID NR. 6, SEQ ID NR. 7, SEQ ID NR. 8 und SEQ ID NR.
9 ist.
16. Verfahren nach Anspruch 1, wobei das Immunogen ein Fusionspolypeptid ist, welches
einen Polyhistidinschwanz zusammengesetzt aus der Aminosäuresequenz ausgewählt aus
der Gruppe bestehend aus SEQ ID NR. 24, codiert durch das Nukleotid SEQ ID NR. 23,
SEQ ID NR. 26, codiert durch SEQ ID NR. 25 und SEQ ID NR. 35 enthält.
17. Verfahren nach Anspruch 1, wobei die Hauptfimbrien-Untereinheit CfaB mit einer Polypeptidsequenz
von SEQ ID NR. 1 codiert durch Nukleotidsequenz SEQ ID NR. 20 ist.
18. Verfahren nach Anspruch 1, wobei die Hauptfimbrien-Untereinheit CsbA mit einer Polypeptidsequenz
von SEQ ID NR. 7 codiert durch Nukleotidsequenz SEQ ID NR. 21 ist.
19. Verfahren nach Anspruch 1, wobei die Hauptfimbrien-Untereinheit CotA mit einer Polypeptidsequenz
von SEQ ID NR. 9 ist.
1. Procédé de production d'une composition pharmaceutique qui est adaptée en tant qu'agent
immunoprophylactique passif contre
Escherichia coli entérotoxigène, le procédé comprenant les étapes suivantes :
a. administration, à un animal non humain domestiqué produisant du lait, d'un immunogène
composé d'une ou de plusieurs adhésines fimbriales d'Escherichia coli de Classe cinq, chacune de la ou des adhésines fimbriales étant liée au niveau de
son extrémité C-terminale à un lieur qui est opérationnellement lié au niveau de son
extrémité C-terminale à une sous-unité fimbriale majeure d'Escherichia coli, dans lequel ledit lieur est composé d'une séquence d'acides aminés sélectionnée dans
le groupe constitué de SEQ ID NO : 10, SEQ ID NO : 12 et SEQ ID NO : 13 ; et
b. collecte du colostrum ou du lait contenant des immunoglobulines anti-adhésines
desdits animaux non humains domestiqués.
2. Procédé selon la revendication 1, dans lequel la concentration en ladite immunoglobuline
anti-adhésine dans ledit colostrum ou ledit lait est ajustée à 0,1 g d'IgG par dose
à 20,0 g d'IgG par dose.
3. Procédé selon la revendication 1, dans lequel ledit animal domestiqué est une vache
ou une chèvre.
4. Procédé selon la revendication 1, dans lequel ladite adhésine fimbriale est sélectionnée
dans le groupe constitué des CfaE, CsfD, CsuD, CooD, CosD, CsdD, CsbD et CotD.
5. Procédé selon la revendication 1, dans lequel ladite sous-unité fimbriale majeure
d'Escherichia coli est sélectionnée dans le groupe constitué des CfAB, CsfA, CsuA1, CsuA2, CooA, CosA,
CsbA, CsdA et CotA.
6. Procédé selon la revendication 4, dans lequel ledit immunogène est un polypeptide
de fusion domaine adhésine fimbriale d'Escherichia coli et queue polyhistidine composé de la séquence d'acides aminés sélectionnée dans le
groupe constitué de SEQ ID NO : 35, SEQ ID NO : 36 et SEQ ID NO : 37.
7. Procédé selon la revendication 1, dans lequel ladite adhésine fimbriale d'Escherichia coli est un monomère ou un polymère de polypeptides d'adhésine.
8. Procédé selon la revendication 1, dans lequel ledit immunogène contient une queue
polyhistidine liée au niveau de l'extrémité C-terminale de ladite sous-unité fimbriale
majeure d'Escherichia coli.
9. Procédé selon la revendication 1, dans lequel ladite adhésine fimbriale est une séquence
d'acides aminés sélectionnée dans le groupe constitué de SEQ ID NO : 11, SEQ ID NO
: 22, SEQ ID NO : 27, SEQ ID NO : 28, SEQ ID NO : 29, SEQ ID NO : 30, SEQ ID NO :
31, SEQ ID NO : 32 ou des fragments de celles-ci.
10. Procédé selon la revendication 4, dans lequel CfaE est composé de la séquence d'acides
aminés de SEQ ID NO : 11 codée par la totalité ou une partie de la séquence nucléotidique
de SEQ ID NO : 18 ou un fragment de celle-ci.
11. Procédé selon la revendication 4, dans lequel ledit CsbD est composé de la séquence
d'acides aminés de SEQ ID NO : 22 codée par la séquence nucléotidique de SEQ ID NO
: 19 ou un fragment de celle-ci.
12. Procédé selon la revendication 4, dans lequel ledit CotD est composé de la séquence
d'acides aminés de SEQ ID NO : 32 ou un fragment de celle-ci.
13. Procédé selon la revendication 1, dans lequel ladite adhésine fimbriale d'Escherichia coli est composée des acides aminés 58 à 185 ou d'une séquence sélectionnée dans le groupe
constitué de SEQ ID NO : 11, SEQ ID NO : 22, SEQ ID NO : 27, SEQ ID NO : 28, SEQ ID
NO : 29, SEQ ID NO : 30, SEQ ID NO : 31, SEQ ID NO : 32.
14. Procédé selon la revendication 1, dans lequel ladite adhésine fimbriale d'Escherichia coli est composée des acides aminés 14 à 205 ou d'une séquence sélectionnée dans le groupe
constitué de SEQ ID NO : 11, SEQ ID NO : 22, SEQ ID NO : 27, SEQ ID NO : 28, SEQ ID
NO : 29, SEQ ID NO : 30, SEQ ID NO : 31, et SEQ ID NO : 32.
15. Procédé selon la revendication 1, dans lequel ladite sous-unité fimbriale majeure
est une séquence d'acides aminés sélectionnée dans le groupe constitué de SEQ ID NO
: 1, SEQ ID NO : 2, SEQ ID NO : 3, SEQ ID NO : 4, SEQ ID NO : 5, SEQ ID NO : 6, SEQ
ID NO : 7, SEQ ID NO : 8, et SEQ ID NO : 9.
16. Procédé selon la revendication 1, dans lequel ledit immunogène est un polypeptide
de fusion contenant une queue polyhistidine composée de la séquence d'acides aminés
sélectionnée dans le groupe constitué de SEQ ID NO : 24 codée par la séquence nucléotidique
SEQ ID NO : 23, SEQ ID NO : 26 codée par SEQ ID NO : 25 et SEQ ID NO : 35.
17. Procédé selon la revendication 1, dans lequel ladite sous-unité fimbriale majeure
est CfaB avec une séquence polypeptidique de SEQ ID NO : 1 codée par la séquence nucléotidique
SEQ ID NO : 20.
18. Procédé selon la revendication 1, dans lequel ladite sous-unité fimbriale majeure
est CsbA avec une séquence polypeptidique de SEQ ID NO : 7 codée par la séquence nucléotidique
SEQ ID NO : 21.
19. Procédé selon la revendication 1, dans lequel ladite sous-unité fimbriale majeure
est CotA avec une séquence polypeptidique de SEQ ID NO : 9.